Navigation Links
miRNA Expression in White Blood Cells

Emmanuel Labourier, Marianna Goldrick, Ambion, Inc.


microRNAs (miRNAs) are endogenous, 21-24 nt RNAs that mediate post-transcriptional gene regulation by pairing with the 3' untranslated region of messenger RNAs and acting as translational repressors. The study of miRNA is an emerging and exciting area of research with applications for basic, applied, and therapeutic science. Recent reports suggest a role for miRNAs in development, cell differentiation, apoptosis, and cancer. Ambion has recently developed several new tools for isolation and detection of miRNAs, which were used to investigate miRNA expression in white blood cells.


Introduction
In the past few years, several discoveries have indicated that miRNAs play a role in various cellular functions. Early studies of miRNAs suggest that they could become a valuable tool for elucidating how gene expression can direct development, differentiation, and proliferation. This area of research could provide more insight into the causes of certain diseases, leading to the identification of novel therapeutic or diagnostic targets. Interest in the identification, detection, and use of small RNA molecules has exploded in response to these discoveries. However, standard RNA isolation and quantitation techniques have been optimized for larger RNAs and are not always favorable for recovering and analyzing smaller RNA species.


Analysis of Small RNA Expression
The size, and in some cases the low expression levels, of small RNAs can make their analysis difficult. The small size of these molecules alone precludes the use of RT-PCR as a detection method, since they are too short to be specifically primed for PCR-mediated amplification. Furthermore, most RNA isolation procedures have been optimized to recover long (>500 nt) messenger RNAs, while ignoring smaller molecules. As a result, conventional RNA extraction methods can result in the loss of substantial amounts of small RNAs from samples. To address the issues of small RNA recovery and detection, Ambion scientists have developed the mirVana line of products, a group of kits designed to provide a complete solution for small RNA analysis.

The mirVana miRNA Isolation Kit (patent pending) was codeveloped with the mirVana miRNA Detection Kit to provide quantitative yields of small RNAs (including miRNA, siRNA, tRNA, and rRNAs) from virtually any biological sample. The patented mirVana miRNA Detection Kit is a set of reagents for small-volume solution hybridization assays that is far more efficient than membrane-based hybridization (e.g. Northern blots). The assay can detect relatively abundant miRNA species, such as miR-124 in brain, in as little as 10 ng of total RNA.

An example of small RNA analysis using these tools is shown in Figure 1. The data show that the same amount of 5.8S and 5S rRNA was recovered from mouse brain, kidney, liver and thymus. In contrast, significant variations in miRNA levels were observed in these tissues. For example, let-7 was more abundant in brain and kidney, and miR-16 was highly expressed in thymus only. As expected, based on published reports [1, 2], miR-124 was detected only in the brain sample.

Figure 1. Analysis of miRNA Expression Across Mouse Tissues. Total RNA was isolated from ~100 mg of the indicated tissues with the mirVana miRNA Isolation Kit. Purified RNA (1 g) was analyzed on a 15% denaturing polyacrylamide gel stained with ethidium bromide (left panel). let-7 miRNA, and 5.8S and 5S rRNA were detected by Northern blot. miR-16 and miR-124 miRNA were detected by solution hybridization with the mirVana miRNA Detection Kit. All probes were labeled and purified with the mirVana Probe & Marker Kit.


Expression of miRNA in Blood Cells
Although miRNAs have been found in all mammalian tissues examined so far, there is little if any published information about miRNA expression in blood. This may be due to the inherent difficulty in isolating high quality RNA from human blood. To examine the efficiency of the mirVana system for use in analysis of miRNAs in white blood cells, blood was collected from 14 healthy volunteers. Leukocytes were fractionated from whole blood and the total RNA population, including miRNA, was extracted using the mirVana miRNA Isolation Kit. The RNA recovered from the fractionated white blood cells was quantitated by UV absorbance and analyzed by ethidium bromide staining on a 15% denaturing polyacrylamide gel. RNA yields ranged from about 20-40 g per 10 ml blood sample. No obvious degradation of the ribosomal RNA and tRNA bands was observed. Four representative samples are presented in Figure 2.

Figure 2. Analysis of miRNA Expression in White Blood Cells. Leukocytes were collected from 10 ml EDTA-anticoagulated blood samples from four different donors, and total RNA was isolated with the mirVana miRNA Isolation Kit. Samples were analyzed as described in Figure 1 except that a probe specific for miR-22 miRNA was used in the solution hybridization assay.


The RNA samples were then analyzed for the presence of several small RNAs using Northern blot or the mirVana miRNA Detection Kit. Interestingly, several miRNAs--let-7, miR-16, and miR-22--were detected in total RNA from white blood cells (Figure 2). miR-16 was the most abundant miRNA in all of the 14 samples. A direct comparison of total RNA samples from other human tissues showed that miR-16 is expressed at a higher level in white blood cells than in any other tissues we have examined (data not shown). While different expression levels were observed across donors for the few miRNA tested, there was no clear donor-specific difference in the global miRNA expression pattern. Experiments are in progress to determine whether other miRNAs may have donor-specific expression patterns and whether these patterns vary over time.


Outlook
As the study of small RNA molecules continues, the need for fast, robust, and sensitive miRNA analysis tools is increasing. Further investigation of the biological role of miRNA will be facilitated by methods such as those incorporated in the mirVana system. High throughput detection of miRNA will undoubtedly help to unravel their complex spatial and temporal expression patterns, as well as determine variations in miRNA levels during tissue development and in disease states.


back to top


Ordering Information
Cat# Product Name Size 1550 mirVana miRNA Probe Construction Kit 30 rxns 1552 mirVana miRNA Detection Kit 100 rxns 1554 mirVana Probe & Marker Kit 30 rxns + 10 marker rxns 1560 mirVana miRNA Isolation Kit up to 40 purifications
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Cancer-Related miRNAs Uncovered by the mirVana miRNA Microarray Platform
2. Detecting miRNA & siRNA
3. Isolate It All: siRNA miRNA Total RNA Native Protein
4. Get the miRNA OutPlant Applications
5. Novel MicroRNA Array Technology for Sensitive miRNA Profiling
6. pSilencer 4.1-CMV: Versatile Vectors for Expression of siRNA, miRNA, and mRNA
7. Precursor miRNAs for Successful miRNA Functional Studies
8. Prepare siRNA and miRNA Probes in Just 1 Hour
9. Reagents for Up and Down-Regulation of miRNA Activity in Mammalian Cells
10. An Epitope Tagging Vector for Gene Expression in Mammalian Cells
11. Antibodies for Studying NMDA Receptor Protein Expression and Synapse-Specific Immunolabeling
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: miRNA Expression White Blood Cells

(Date:7/10/2014)... (PRWEB) July 10, 2014 OMICS Group’s ... techniques during 18-20 August, 2014 at Double Tree by ... the recent developments in Analytical & Bioanalytical research methods ... , Speaking on this occasion, Dr. Srinubabu Gedela, MD ... Acta conference is a remarkable one in bringing a ...
(Date:7/10/2014)... -- Kainos Capital, a firm specializing in acquiring and managing ... acquired the Slim-Fast brand from Unilever. Unilever will retain ... transaction were not disclosed. Slim-Fast is a ... ready-to-drink shakes, powders, bars and snacks to retail customers ... the United Kingdom and Ireland.  ...
(Date:7/10/2014)... the fight against terrorism with the creation of a ... the use of light and special glass fibres. , ... Chemical , the researchers describe a novel optical fibre ... as 6.3 ppm (parts per million). It requires an ... explosives detection has involved looking for metals that encase ...
(Date:7/10/2014)... that power our laptops and electric vehicles could store ... with the help of a sponge-like silicon material. ... graphite traditionally used in one of the battery,s electrodes, ... storage capacity of graphite. A paper describing the material,s ... in Nature Communications . , "Silicon has long ...
Breaking Biology Technology:A Critical Review of Analytical Techniques for Improved Living Standards 2A Critical Review of Analytical Techniques for Improved Living Standards 3A Critical Review of Analytical Techniques for Improved Living Standards 4Kainos Capital Acquires Slim-Fast From Unilever 2Detecting trace amounts of explosives with light 2Silicon sponge improves lithium-ion battery performance 2Silicon sponge improves lithium-ion battery performance 3
... , , , ... Board: SYMD), a biomaterials company engaged in the development ... the company,s bioresorbable adhesion barrier film for the reduction ... the Brazilian Ministry of Health for use in all ...
... DURHAM, N.C., Sept. 9 Oxygen Biotherapeutics, Inc. (OTC Bulletin Board: ... asks shareholders to approve a 1-for-15 reverse stock split at the ... "At last years, general annual meeting we explained the problems of ... promised to seek a listing change as soon as we can ...
... MUMBAI, India, September 9 , ... its First Domain-Specific Learning,and Consulting Solutions for Health Care ... Interactive Systems (TIS)-has,recently announced the launch of its first ... industry. TIS formally,launched this initiative with the appointment of ...
Cached Biology Technology:SyntheMed Receives Brazilian Regulatory Approval for REPEL-CV(TM) 2Oxygen Biotherapeutics, Inc. Seeks Shareholder Approval for Reverse Stock Split 2Tata Interactive Systems Initiates Vertical-Focused Learning Solutions for Health Care, Pharmaceutical, and Biotechnology 2Tata Interactive Systems Initiates Vertical-Focused Learning Solutions for Health Care, Pharmaceutical, and Biotechnology 3Tata Interactive Systems Initiates Vertical-Focused Learning Solutions for Health Care, Pharmaceutical, and Biotechnology 4
(Date:7/10/2014)... ALBANY, New York , July 7, 2014 /PRNewswire/ ... published by Transparency Market Research "Electronic Access Control Systems ... Analysis, 2014 - 2019," the global Electronic Access Control ... 2013 and is expected to grow at a CAGR ... estimated value of USD 31,187.8 million in 2019. ...
(Date:7/10/2014)... 2014  Acuity Market Intelligence today released forecasts from "The ... that the global market for National Electronic ID (eID) programs ... During this time, the number of National eID cards in ... Asia , with its vast population, will dominate ... issued, while Europe trails a distant ...
(Date:7/10/2014)... genus Axinaea belongs to the large, mainly tropical ... ca. 5000 species in the family rely on bees for ... by other insects or vertebrates. The flowers of Axinaea ... flowers and the petals of the different species are pink, ... out based on the contrasting colours of their bulbous appendages. ...
Breaking Biology News(10 mins):Electronic Access Control Systems Market is Expected to Reach USD 31.2 Billion Globally in 2019: Transparency Market Research 2Electronic Access Control Systems Market is Expected to Reach USD 31.2 Billion Globally in 2019: Transparency Market Research 3Electronic Access Control Systems Market is Expected to Reach USD 31.2 Billion Globally in 2019: Transparency Market Research 4Electronic Access Control Systems Market is Expected to Reach USD 31.2 Billion Globally in 2019: Transparency Market Research 5National Electronic ID Programs Generate $54 Billion Between 2013 and 2018 with 3.5 Billion National eID Card Holders Worldwide 2Novel type of bird pollination mechanism discovered in South America 2
... detection methods, researchers at the Saint Louis University Institute ... by which the HIV virus infects, or integrates, healthy ... for HIV. , Although scientists theorized that two ends ... healthy cell in order to infect it, until now, ...
... like other biological attributes, is shaped by evolution ... species that are in other ways very similar ... the visual world slightly differently, using photoreceptors that ... studying a special group of closely-related fish species ...
... stroke has taken a major step forward following promising results ... of Manchester have shown in laboratory studies that a naturally ... death. , The team, led by Professor Nancy Rothwell and ... first small trial of IL-1ra in patients, which are published ...
Cached Biology News:SLU researchers uncover direct evidence on how HIV invades healthy cells 2Multiple genes permit closely related fish species to mix and match their color vision 2Stroke treatment a step closer after trial 2
... is the major proteolytic system in ... it catalyzes the selective degradation of ... elimination of proteins with abnormal conformation ... The critical protease in this pathway ...
... [1320] to RFC1 Immunogen Recombinant protein ... Factor C expressed in E. coli. Reactivity ... cerevisiae. Not yet tested in other species. This ... Background Information Replication factor C is ...
... Bacterial DNA Isolation Kit provides a rapid ... genomic DNA from the various bacterial species ... the isolation of genomic DNA from both ... milk samples. ,Purification is based on ...
... Chemicon Select Tissue Arrays are intended for ... tissues were fixed in neutral buffered formalin ... and diagnosis was performed by certified pathologists. ... paraffin blocks and relocated into a new ...
Biology Products: